Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disease that currently has no cure. ALS inexorably progresses to paralysis and to death that usually occurs within five years of diagnosis. None of the available treatments appreciably prolongs life or improves quality of life. A recent advance in ALS research was the finding that the TDP gene product, TDP-43, is a major protein component of ubiquitin-positive inclusions, which are a hallmark of sporadic ALS and frontotemporal lobe degeneration (FTLD). When frustration haunts TDP-43 for a while, a breakthrough comes up with the discovery of mutation in the TDP gene in ALS. A critical step in understanding a disease gene is determining the nature of the pathogenic gene mutation. Such pathogenic mutations may cause a gain of function, a loss of function, or a dominant negative effect. With great uncertainty, current studies suggest that both loss of function and gain of function possibly contribute to the pathogenesis of the TDP gene mutation. Overexpression of the human form of the pathogenically mutated TDP gene induces ALS in transgenic mice and rats. However, transgenic studies are unable to differentiate whether a mutant gene causes disease by gain of function or by dominant negative effect because both types of mutation can induce disease in transgenic animals. Therefore, a more sophisticated model is required for determining the nature of the TDP gene mutation. We have created conditional TDP knockin mice in which the mouse TDP gene is replaced by the normal or mutant human TDP gene flanked by loxP sites, allowing the TDP knockin genes to be deleted by Cre-mediated recombination. We have also established neuronal cultures derived from TDP knockin mouse embryos. Using TDP knockin mice and primary neurons as two complementary model systems, we will unequivocally determine whether pathogenic mutation of the TDP gene causes ALS by a gain of function, a loss of function, or a dominant negative effect. Findings from these studies will provide a foundation for mechanistic studies of the ALS. The resulting knockin mice will be far more physiologically relevant than any of the ALS models currently available and will be ideal for testing potential ALS therapies. PUBLIC HEALTH RELEVANCE: Using conditional TDP knockin mice as models, this application will dissect pathogenic mechanisms underlying amyotrophic lateral sclerosis and will provide a foundation for development of therapies for this fatal disease.